Nick Konkol: Hi. I think I should take a second here to tell you a little bit about Professor Mitchell’s laboratory, the laboratory that I work in. It’s really an applied microbiology or a general microbiology laboratory, but the focus has been the interaction of microbes with surfaces. That’s kind of afforded us the opportunity to work with a lot of different cultural heritage materials. As you probably know, microbial growth can be a factor in the deterioration of cemetery materials.

Over the years we’ve studied a lot of different projects that have dealt with microbes and their interactions with cultural heritage materials. We’ve worked on corrosion in the Arizona memorial in Pearl Harbor. We’ve worked on investigating the types of bacterial communities that live on surfaces and within stone that has been used to build the Mayan monuments and their cities. We’ve also looked at mold that has contaminated the Apollo spacesuits, so the spacesuits that the astronauts wore to the moon. Those are held in the Smithsonian.

Since I’ve been at the lab, we’ve kind of looked at more general projects, one of them being this early detection of mold growth, and another project that involves the de-colorization of pigments. This has to do with probably some red staining that some of you are probably familiar with on marble monuments and structures. So I’ll talk about those today.

marble magnified 500X with thin filaments of mold and some of the reproductive structures

The first deals with early detection of mold growth. In listening to the problems you guys are having, I don’t know how much relevance this has. You’re dealing with people backing over your monuments with their lawnmowers and so I just don’t … I hope you guys see some value in this. If you have a monument that looks like this, this is kind of a black melanin containing fungus or mold that’s growing on the surface of this piece of marble. If you have something like this, our assay really isn’t going to help you. I kind of look at it as an early detection device. This is a piece of marble magnified 500X. You can see some very thin filaments of mold and some of the reproductive structures here. This is when you would want to use the type of assay that I’m about to tell you about. But I can envision some other uses sitting here listening to you guys. Maybe to evaluate some of your remediation efforts you might be able to use an assay like this, or to maybe keep track of your maintenance, this assay might be useful for something like that. I’ll leave it up to you to come up with an answer to that.

So they’re working on this assay and it has to do, or it utilizes the fact that mold cell walls are made of the rigid polymer chitin. You might be familiar with chitin. It makes up exoskeletons of insects, crustaceans, it’s a really great molecule. If you’re a lobster and you have a nice chitin exoskeleton you are very well protected from predators. The problem with having a chitin exoskeleton is that every now and then, if you’re growing, you’ve got to get rid of it. It’s not going to grow with you. So, if you’re a lobster you shed your chitin shell, you’re very susceptible to predation during those days and weeks as you’re rebuilding your chitinous shell.

Now fungi, they’re molds, they kind of have the same problem. They do have to break their cell wall in order to grow. They don’t molt like a crustacean would. They continuously break it by producing an enzyme called chitinase. So they keep breaking their cell wall and they grow wherever they break their cell wall. So they can continuously grow by producing this chitinase. So we use a chitin molecule that has a fluorescent tag attached to it and we use that to detect growing mold. The growing mold is going to be producing chitinase, and if we have this molecule that is broken down by chitinase and fluoresces, we can read that fluorescence. We call it MUF, and that’s an abbreviation for 4-methyl-umbelliferone. That’s the molecule that does the fluorescing.

I’ve got a little animation here to kind of show you how this assay works. We have a molecule of chitin bound to this MUF molecule, and in this state the MUF molecule does not fluoresce. It doesn’t have any activity in this situation. But when it’s broken that MUF molecule will fluoresce. When you introduce a chitinase that’s being produced by a mold, it will break that bond, the MUF is separated from the chitin, and it will begin to fluoresce. The more chitinase you have, the more
fluorescence you will get when you’re looking at the fluorescence.

Conducting the assay

So running the assay is very, very simple. I think it’s great for conservators, librarians, archivists, people who deal with cultural heritage materials. We go out and you just collect a sample using a cotton swab. You might want to dip it in some distilled water and then you want to sample a uniform area every time you do this. So here I’m showing you a sample that’s being take from a fungus that’s growing on a nutrient rich plate, so there’s lots of fungus there and we’ve cut out a one centimeter square area there. Kind of like a stencil that you would sample every single time. I would imagine on a cemetery material you’d probably want to sample a larger area. But yeah, it’s easy, it’s just going out and swabbing an area. Then you take that swab and you put it into a test tube with just a few chemicals. You could take these out to a cemetery and mix everything there on site if you really wanted to. You just let things run for about a half an hour. Then you have to read the fluorescence that is coming out of your sample. There are portable, handheld fluoremeters that you could take with you out into the cemetery. We use a more substantial unit, it gives you a lot more sensitivity and reproduce-ability in your results.

When we started doing this work we wanted to establish a relationship between the fluorescence values that we got out of our fluoremeter and the amount of mold that was actually present on the surfaces that we were looking at. We noticed that when we measured the fluorescence of known amounts of fungal biomass we got a very nice linear straight line relationship between the amount of fluorescence that was generated and the amount of biomass that we were assaying. If we look at this area of the graph, you can see just how small an amount of mold we can actually detect with this. This is about two micro-grams of mold that we can detect in a pretty small sample. So it’s a very, very sensitive assay.

Early detection of mold on marble

We performed the assay on some marble coupons that we had obtained and we measured it over time, took a sample very early on to kind of establish our baseline. If you were going to perform this assay yourself, we would want to get some negative control samples so you know what the background fluorescence level is. Over time, you can see that the amount of fluorescence increased which meant that the amount of fungal biomass increased over time. You can see that after about seventy-two hours we had an amount of, or we could read an amount of biomass, that was statistically different than the previous time points. Of course after more time, after ninety-six hours, the fungus was actually visible on the marble at this point, we detected a very large, large amount. So we established that we could actually use this assay on marble.

So we conclude that we can detect as little as two micro-grams of mold and that we can detect it before it’s visible. So on this previous slide here it really wasn’t visible until a little after seventy-two hours. So we were basically detecting an invisible amount of mold on this marble. So it could be pretty handy and we detect it on a variety of materials. Marble, concrete, wood, and a variety of other cultural heritage materials if you guys are interested. Paper, metals, textiles, canvas, it’s a very malleable assay. You can use it on a wide variety of surfaces. So that kind of wraps up our fungal detection part of the talk.

The next part is de-colorization of pigments. We wanted to demonstrate the presence of pigment producing microorganisms within a stained marble sample. We wanted to investigate the potential of an enzyme called laccase to de-colorize pigments isolated from the surface of Isamu Noguchi’s ‘Slide Mantra’. Laccase is an enzyme that comes from, I believe it’s the soybean plant. Yeah, I think it’s the soybean plant. Then there’s a variety of other enzymes out there that come from different fungi and other organisms that actually do the same thing as laccase. We tested those as well but I’m just going to kind if share the laccase data with you here today.

‘Slide Mantra’, it is located in Bayfront Park, Miami. It was assembled in 1991. It’s three meters tall, weighs twenty-nine tons, and is eight different blocks of marble that have all been dry-mounted together. This is kind of an important point because there’s a little bit of controversy going on in the field of red stained marble as to whether or not the red stains that you’re looking at are chemical in nature or if they’re biological in nature. If lead is present it’s very possible that the red staining that you’re seeing is different lead oxides that have formed from the lead that was used in the construction of whatever object you’re looking at. But this particular piece, it doesn’t have, or it didn’t I should say, have any lead pins or spacers or any lead associated with it whatsoever. It was just a big chunk of marble. So no lead oxides, we’re assuming no lead oxides were present here, and … Oh and I should say it’s called ‘Slide’ because there’s a big hole in the back that a person can go in through and then there’s a staircase, you can see the top of the staircase right here, that you can walk up, and then you can slide down, so ‘Slide’. He’s got a couple pieces like this that are around the world.

Okay, so the piece underwent a very expensive restoration a few years back. It was, as I said, it’s dry mounted, it’s down in Florida, lots of hurricanes in Florida. Water had infiltrated the cracks in between all the marble blocks, and I think it was Hurricane Wilma that came by, lots of lateral winds, the water in between the cracks cut down all the friction and the statue just … The piece just fell apart, so it was time to do some remediation. Prior to that, a red/reddish brown staining had been building and building in the surface of this piece. After the remediation process they were able to get rid of most of the staining. They used D2 and repeated steam treatments. The D2 did a really good job of killing all the microorganisms, didn’t do so good of a job eliminating the stain. The steam didn’t help so much either. A little bit of the red stain still exists on this particular piece. So we kind of wanted to investigate this stain.

The pigment resembles prodigiosin

Some people at the company that we work with, they went out … The people who did the restoration. They went out, they took some swabs for us, and we found a pigmented bacterium was associated with the stained areas of the piece. Places on the piece that they had swabbed that weren’t red stained we did not get this red stained bacterium out of. We were able to use some molecular techniques to identify it as Serratia Marcescens. Serratia Marcescens, it’s an interesting organism. Some people think that this might be attributed … People claiming that statues were bleeding, they think it might be because of this particular organism. The red stain from S. Marcescens, it’s attributed to a pigment called prodigiosin. We wanted to see if the organism that we isolated is producing prodigiosin. So I went into Professor Mitchell’s freezer, and wouldn’t you know it, we had a strain of S. Marcescens in there. I grew it up, I extracted the pigments, and I looked at the infrared spectra of the S Marcescens extract and we got this nice characteristic fingerprint of prodigiosin. Then I grew up our isolated bacteria from ‘Slide Mantra’ and I ran that up against it and it’s basically identical. So we’re probably dealing with a prodigiosin here.

Now de-colorization reactions. Enzymes like laccase, they’ll take a dye and oxidize it and the resulting products will be de-colorized. So I took our extracted pigment, this is the pigment I extracted from the bacteria that was growing on ‘Slide Mantra’, I measured its absorbance, it has a maximum right here, I think it’s around five hundred and seventy. The de-colorized product, once it’s been exposed to laccase, you can see that the peak disappears. If we plot this out over time, you can see our no laccase control retains its nice pink, it’s got kind of a pinkish hue to it when we extract it from the organism. Whereas, the sample that exposed to laccase, you can see the absorbance dropped and the product is de-colorized. So it worked very nicely when we extracted it from the bacterium.

To conclude, we successfully isolated S. Marcescens from the stained areas of ‘Slide Mantra’ and the pigments extracted could be de-colorized with the enzyme laccase. Like I said, we used some other enzymes as well, we used lipoxidase, manganese peroxidase, and they worked pretty well too. They have some issues though. If you expose lipoxidase to air it will hurt its activity so we kind of went with laccase because it’s something we could actually envision someone, someday, taking out into the field and using a headstone, something like that. Future work is going to involve testing this method in the field. We don’t have access to ‘Slide Mantra’ anymore. They had access to it because they were restoring it and we’re not allowed to touch it anymore. We’re kind of looking for new field work that we could actually try this on.

Speaker 2: Oh great I have a whole cemetery full of stuff like that. When do you want to visit?
Nick Konkol: Something that doesn’t have any lead. That’s kind of what we’re looking for.
I just wanted to mention, Professor Ralph Mitchell, his laboratory is doing this work. Another post doc in the lab, Chris McNamara, he helped with a lot of this work as well. The company that we were working with on ‘Slide Mantra’, Conservation Solutions, they provided us with samples and a little bit of our funding to do this kind of work. And then people at the Strauss Center For Conservation at Harvard. They were very generous, they performed our IR Spectros for free, it was very nice of them. They did the analysis, so it was very nice. We were able to do this on a budget and get some…

Abstract:

Microbial growth is frequently a factor in the deterioration of stone cemetery materials, particularly in damp or humid environments. Metabolically diverse bacteria and fungi attack the stone statuary, monuments and facades through enzymatic activity and through mechanical weathering. The detection and elimination of these growths can be difficult. Removal of their residues is even more difficult as the organisms are frequently embedded deep within fissures and surface crevices. Recently, microbiologists and conservators have begun using enzymes both to safeguard and restore stone artifacts. We have developed two new enzyme-based techniques that can be applied to cemetery materials.

Mold detection
Protecting outdoor stonework from mold is a challenge to conservators. Molds are ubiquitous components of the air we breathe and have the capacity to infect a variety of materials. These fungal infections are often overlooked until they attain enough biomass to be visible to the human eye. A great deal of damage can be done in the time between infection and detection. Current methods used for detecting and measuring fungal biomass on surfaces, such as microscopic biovolume estimates and chemical analysis of the mold, are expensive and time-consuming. In order to facilitate early fungal detection, we have developed a rapid, inexpensive, and non-destructive means of identifying fungal growth.

Fungal cell walls are composed of the rigid biopolymer chitin that fungi must continually deconstruct and rebuild in order to grow. Deconstruction of chitin is carried out with chitinase, an enzyme that catalyzes the break-down of N-acetyl-β-D-glucosamine (NAG). Fluorogenic 4-methylumbelliferyl (MUF)-labeled NAG can be used as a substrate to detect chitinase activity during mold growth. Originally developed to detect and quantify fungal chitinase activity in soil1 it was quickly demonstrated that MUF-NAG could be used to detect fungi on building surfaces2. We used this assay to quantify fungal biomass on a variety of materials, including marble with sensitivity comparable to that of chemical techniques (lower detection limit near 40 μg dried mycelium). Our assay can be used to detect early mold growth on stone surfaces as an aid to conservators in identifying “at risk” materials.

Stain removal
Remediation of outdoor sculptures, monuments and facades that have suffered from microbial deterioration also poses a challenge to conservators. Treatments developed to clean them are often time-consuming, expensive, risk further damage, and are not always effective. Enzymes may overcome many of these limitations, and have already found use in the remediation of frescoes and easel paintings3. We have begun to expand the use of enzymes for remediation by conducting experiments on open-air marble sculptures. Many marble sculptures, monuments, and facades throughout Europe and North America suffer from biodeterioration. It often manifests itself as unsightly red stains whose removal has been problematic.

We determined that red-brown stains on Isamu Noguchi’s marble sculpture Slide Mantra in Bayfront Park, Miami, Fl, were probably caused by pigment-producing microorganisms. Traditional cell culture methods were first used to isolate a red-pigmented bacterium from a stained area of Slide Mantra. Sequencing and analysis of the 16S rRNA gene identified the organism as a strain of Serratia marcescens. Fourier transform infrared spectroscopy demonstrated that the pigment produced by the Bacteria was most likely a prodigiosin. Our analysis found that the enzyme laccase, isolated from the fungus Trametes versicolor, effectively decolorized pure prodigiosin (Fig 2). Tests of the effects of this enzyme on the bacterium that stained Slide Mantra similarly decolorized the stain. This study suggests that enzymatic decolorization may be applicable to stains on culturally significant marble caused by microbial colonization. We are currently investigating the use of this enzyme and others to remediate the surfaces of Slide Mantra and other marble sculptures exposed to outdoor air.